Methods for Screening Proteins Using DNA Encoded Chemical Libraries as Templates for Enzyme Catalysis

• US 20150361420A1
• Filed: 08/31/2015
• Published: 12/17/2015
• Est. Priority Date: 03/21/2012
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Hudson, Michael E., Jacobson, Joseph
• Granted Patent: US 10,308,931 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays

Abstract

Disclosed are methods, compositions and devices for screening a protein library for proteins having a desired activity, such as capable of catalyzing the formation of a bond between two reactants. In an exemplary embodiments, a plurality of proteins are expressed in vitro from a plurality of nucleic acids, the plurality of proteins are exposed with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore, the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes is detected and the complexes showing detectable fluorescence energy transfer are isolated, thereby isolating proteins having the desired enzymatic activity.

Claims

1. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) providing, at distinct features of a solid support, a plurality of nucleic acids, each nucleic acid having a predefined sequence;

b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;

c) exposing the plurality of proteins to two single-stranded oligonucleotides having complementary sequences, each single-stranded oligonucleotide having a reactant and a fluorophore;

d) detecting fluorescence of a complex having a protein having the desired activity, the reactant, the single-stranded oligonucleotide and the fluorophore; and

e) isolating the complex showing detectable fluorescence energy transfer, thereby isolating the protein having the desired activity.

2. The method of claim 1 wherein the desired activity comprises formation of a bond between two reactants.

3. The method of claim 1 wherein the protein having the desired activity specifically binds two reactants.

4. The method of claim 1 wherein the reactant is at one end of the single-stranded oligonucleotide and the fluorophore is at the other end of the single-stranded oligonucleotide.

5. The method of claim 1 wherein each distinct feature is a well of a microwell plate.

6. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) providing, at distinct features of a solid support, a plurality of nucleic acids, each nucleic acid having a predefined sequence;

b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;

c) exposing the plurality of proteins to a first and a second oligonucleotide each, at one end, having a first and a second reactant, respectively, wherein a protein having a desired activity binds to the first and second reactants, thereby forming a complex; and

d) ligating free ends of the first and second oligonucleotides complexed with the protein having the desired activity.

7. The method of claim 6 wherein the first and second oligonucleotides are double-stranded oligonucleotides.

8. The method of claim 6 wherein the first and second oligonucleotides are single-stranded oligonucleotides.

9. The method of claim 8 further comprising adding a helper oligonucleotide capable of bridging the single-stranded oligonucleotides bound to the protein having the desired activity.

10. The method of claim 6 wherein each distinct feature is a well of a microwell plate.

11. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) culturing a host cell comprising a first nucleic acid sequence encoding a protein fused to aga-2 and a second nucleic acid sequence encoding a transcription activator-like effector fused with aga-1, so as to express the protein and the transcription activator-like effector as a first complex at the cell surface;

b) exposing the first complex to a first single-stranded oligonucleotide having a first reactant, wherein the first single-stranded oligonucleotide binds to the transcription activator-like effector to form a second complex;

c) exposing the second complex to a second single-stranded oligonucleotide having a second reactant and a detectable label, wherein the second single-stranded oligonucleotide has a sequence complementary to the first single-stranded oligonucleotide; and

d) detecting the detectable label at the cell surface, the presence of which indicates a protein having a desired activity.

12. The method of claim 11 wherein the host cell is a yeast cell.

13. The method of claim 11 further comprising isolating the host cell expressing the protein having the desired activity.

14. The method of claim 11 comprising culturing a plurality of host cells expressing a library of proteins.

15. The method of claim 14 wherein the library of proteins comprise a library of protein variants.